MiRNAs as novel therapeutic targets and diagnostic biomarkers for parkinsons disease

ABSTRACT

The disclosure provides pharmaceutical compositions including an oligonucleotide that down-regulates the over-expression of at least one miRNA of SEQ ID NOs: 1-283. The oligonucleotide may be complementary to the nucleotide sequence of at least one of SEQ ID NOs: 1-283, or hybridizes under stringent conditions to a nucleotide sequence of at least one of SEQ ID NOs: 1-283. Further provided are methods of diagnosing Parkinson&#39;s Disease (PD) in a subject. The methods may include detecting the level of expression of at least one miRNA of SEQ ID NOs: 1-283 in a biological sample from the subject, and comparing the level of expression in the sample to the level of expression in a reference. Further provided are methods for treating, preventing, or reducing the risk of PD. Kits are also provided.

This application claims the benefit under 35 U.S.C. §371 ofInternational Application No. PCT/US2013/051849, filed Jul. 24, 2013,which claims the benefit of U.S. Provisional Application No. 61/675,603,filed Jul. 25, 2012, which are incorporated by reference herein in theirentirety.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/675,603, filed Jul. 25, 2012, which is incorporated by referenceherein in its entirety.

FIELD

This disclosure relates to therapeutic and diagnostic markers forParkinson's Disease.

INTRODUCTION

MicroRNAs (miRNAs) are small, non-coding, regulatory RNAs of 18-24nucleotides in length. Mature miRNAs regulate messenger RNAs (mRNAs) oftheir downstream target genes by base-pairing to their target sites tospecify cleavage of the target mRNAs or to destabilize the mRNA and/orrepress translation of the targeted mRNA. miRNAs are expressed in thenervous system and modulate large numbers of neuronal genes, playingimportant roles in neurogenesis and neurodegenerative diseases. Besidesbeing a major level of gene expression regulation, miRNAs also arerecognized as excellent biomarkers for various diseases, especially inthe diagnosis and prognosis of cancer. However, the roles of miRNAs inthe pathogenesis of Parkinson's Disease and their potential asbiomarkers for Parkinson's Disease have not being fully studied.

SUMMARY

In some aspects, the disclosure relates to pharmaceutical compositionsincluding an oligonucleotide that down-regulates the over-expression ofat least one miRNA of SEQ ID NOs: 1-283. The oligonucleotide may be a)complementary to the nucleotide sequence of at least one of SEQ ID NOs:1-283, or b) hybridizes under stringent conditions to a nucleotidesequence of at least one of SEQ ID NOs: 1-283.

In further aspects, the disclosure relates to methods of diagnosingParkinson's Disease (PD) in a subject. The methods may include detectingthe level of expression of at least one miRNA of SEQ ID NOs: 1-283 in abiological sample from the subject, and comparing the level ofexpression in the sample to the level of expression in a reference. Anincreased or decreased level of expression in the sample compared to thelevel of expression in the reference may identify the subject as havingPD or who is at risk of developing PD. In some aspects, a miRNAsignature may be used to diagnose a subject at risk for developing orhaving PD.

Another aspect of the disclosure provides methods for treating,preventing, or reducing the risk of PD associated with aberrantexpression of a miRNA in a cell, tissue, or animal, the methodcomprising contacting the cell, tissue, or animal with a pharmaceuticalcomposition including an oligonucleotide that down-regulates theover-expression of at least one miRNA of SEQ ID NOs: 1-283. Theoligonucleotide may be a) complementary to the nucleotide sequence of atleast one of SEQ ID NOs: 1-283, or b) hybridizes under stringentconditions to a nucleotide sequence of at least one of SEQ ID NOs:1-283.

In yet another aspect of the disclosure, kits are provided forperforming measurement of a miRNA signature, the miRNA signaturecomprising at least one miRNA of SEQ ID NOs: 1-283, wherein the kitcomprises reagents for measuring an expression level of at least onemiRNA of SEQ ID NOs: 1-283.

The disclosure provides for other aspects and embodiments that will beapparent in light of the following detailed description and accompanyingFigures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a heat-map of miRNAs differentially expressed in the putamenby clinical diagnosis.

FIG. 2 is a heat-map of miRNAs differentially expressed in the putamenby unified staging system for Lewy-body disorders (LBDs).

FIG. 3 is a heat-map of miRNAs differentially expressed in cerebrospinalfluid (CSF) by clinical diagnosis.

FIG. 4 is a heat-map of miRNAs differentially expressed in CSF byunified staging system of LBDs.

FIG. 5 is a diagram of miRNA signatures that may be used to reflect theonset of Lewy body (LB) pathology.

FIG. 6 is a diagram of miRNA signatures that may be used to reflect theonset of LB pathology.

FIG. 7 is a diagram of miRNA signatures that may be used to reflect theonset of PD.

FIG. 8 is a diagram of miRNA signatures that may be used to reflect theonset of PD.

FIG. 9 is a diagram of miRNA signatures that may be used to reflect theonset of cognitive impairment in PD.

FIG. 10 is a diagram of the genomic organization of miR-212/132 in thefirst intron of a non-coding RNA gene on human Chr1p13.3.

FIGS. 11A-D are graphs of the expression of miR-132 and miR-212.

DETAILED DESCRIPTION

In a broad sense, the disclosure relates to therapeutic and diagnosticmarkers for Parkinson's Disease (PD). The present invention relates tothe discovery of differential expression levels of various miRNAs in PDcompared to normal tissue.

miRNAs are newly recognized, small, regulatory RNAs, which regulate geneexpression by repressing translation and/or break down mRNAs of theirdownstream target genes. Misregulation of miRNAs in the central nervoussystem contributes to neurodegenerative disorders. To identify miRNAsinvolved in the pathogenesis of PD, fresh frozen putamen andcerebrospinal fluid (CSF) samples of 10 normal control, 12 (ILBD), 10PD, and 10 PD with dementia but no Alzheimer's disease (AD) (PDDnoAD)cases were obtained from the Sun Health Research Institute (SHRI) (Table1 and Table 2). By Unified Staging System for Lewy body diseases (LBDs),besides 9 normal controls, these samples include 12 Stage 2, 9 Stage 3,and 11 Stage 4 samples, representing increasing severity of thediseases.

TABLE 1 Subjects Classified by Clinical Diagnosis and Unified StagingSystem for LBDs (USSLBD) Stages Diagnosis Stage 0 Stage 2 Stage 3 Stage4 total Control 9 9 ILBD 7 5 12 PD 3 2 5 10 PDD no AD 2 2 6 10 Total 912 9 11 41 Beach T G et al. Unified staging system for Lewy bodydisorders (LBDs): correlation with nigrostriatal degeneration, cognitiveimpairment and motor dysfunction. Acta Neuropathol. 2009, 117; 613-634.Classification of subjects with LBDs by Lewy-type α-synucleinopathy(LTS) distributions: Stage 0: No LTS. Control group. Stage 1: Olfactorybulb only; Stage 2: Brainstem or Limbic predominant; Stage 3: Brainstemand Limbic; Stage 4: Neocortical.

TABLE 2 Main basic characteristics of the study subjects classified byclinical diagnosis. SN pigmented α-Synuclein gender ApoE-s4 PMI neurondensity score No. age* (% M) (%) (mean +/− SD) MMSE** UPDRS*** lossscore**** (average +/− SD) control 9 82.1 +/− 6.8 60 22.0 2.4 +/− 0.629.0 (1.3) 7.0 (5.4) none (6), mild (3) 0 (73-97) (9) (9) ILBD 12  86.3+/− 4.85 67 16.7 2.7 +/− 0.6 28.0 (1.7) 7.0 (5.1) none (5), mild (3), 8.6 +/− 8.1 (73-91) (8) (7) mod (1), severe (3) PD 10 79.4 +/− 5.9 5518.2 6.2 +/− 4.2 26.8 (3.3)  9.6 (14.9) mile (1), mod (2), 18.0 +/− 5.3(72-90) (4) (6) severe (8) PDD 10 76.1 +/− 5.9 60 20.0 3.0 +/− 1.4  17.1(10.0) 25.3 (39.3) none (1), severe (9) 19.1 +/− 5.2 (no AD) (69-84) (7)(6) *age is presented by average +/− standard deviation (range); **MMSEis presented by, average (standard deviation) (number of objects withMMSE data); ***UPDRS is presented by average (standard deviation)(number of objects with MMSE data), and was obtained from subjects whowere in the “off state” with respect to dopaminergic therapeuticeffects.

Subsequently, miRNA-proof total RNA from all putamen and CSF samples wasisolated using a mirVana™ miRNA isolation system (Life Technologies). Onaverage, 0.69+/−0.06 μg of total RNA/mg putamen tissue (n=41); and2.74+/−0.21 μg of total RNA/mL CSF samples (n=41) was obtained. Therewas no significant difference in quantity of total RNA isolated fromdifferent diagnostic groups. Subsequently, miRNA profiling in allputamen (n=41) and CSF total RNA (n=41) was performed using the TaqmanmiRNA microarrays (Applied Biosystem), which detected 754 known humanmiRNAs in total, to establish miRNA transcriptomes of the putamen andCSF of all 41 cases.

By comparison of these miRNA-expression profiles, miRNAs differentiallyexpressed in the putamen and CSF among different groups classified byclinical diagnosis, by Unified Staging System for LBD, and by the extentof Substantia Nigra (SN) neuron depigmentation were identifed. Asfurther detailed in the Examples, expression profiles were analyzedusing a two-tailed t test between two groups. Significant differentialexpression was identified as p<0.05 and ≧2 fold changes, including bothincreased and decreased expression levels by ≧2 folds. Exemplary miRNAs(SEQ ID NOs: 1-283) are shown in Table 39. In some embodiments, at leastone miRNA selected from SEQ ID NOs: 1-283 may have increased ordecreased expression relative to a reference, control, or normal sampleby at least about 1.5-fold, at least about 1.6-fold, at least about1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at leastabout 2.0-fold, at least about 2.1-fold, at least about 2.2-fold, atleast about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold,at least about 2.6-fold, at least about 2.7-fold, at least about2.8-fold, at least about 2.9-fold, at least about 3.0-fold, at leastabout 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, atleast about 3.4-fold, at least about 3.5-fold, at least about 3.6-fold,at least about 3.7-fold, at least about 3.8-fold, at least about3.9-fold, or at least about 4.0-fold.

TABLE 39 Sequences of exemplary miRNAs. SEQ miRNA Sequences ID NOmiRNA signature hsa_miR_590_3P_002677 UAAUUUUAUGUAUAAGCUAGU 1distinguishing hsa_miR_213_000516 ACCAUCGACCGUUGAUUGUACC 2 control vs PDhsa_miR_9#_002231 AUAAAGCUAGAUAACCGAAAGU 3 hsa_miR_191#_002678GCUGCGCUUGGAUUUCGUCCCC 4 hsa_miR_497_001043 CAGCAGCACACUGUGGUUUGU 5hsa_miR_664_002897 UAUUCAUUUAUCCCCAGCCUACA 6 hsa_miR_99a#_002141CAAGCUCGCUUCUAUGGGUCUG 7 hsa_miR_1183_002841 CACUGUAGGUGAUGGUGAGAGUGGGCA8 hsa_miR_340#_002259 UCCGUCUCAGUUACUUUAUAGC 9 hsa_miR_628_3p_002434UCUAGUAAGAGUGGCAGUCGA 10 rno_miR_7#_001338 CAACAAAUCACAGUCUGCCAUA 11hsa_miR_29a#_002447 ACUGAUUUCUUUUGGUGUUCAG 12 hsa_miR_142_3p_4373136UGUAGUGUUUCCUACUUUAUGGA 13 miRNA signature hsa-miR-590-3P-002677UAAUUUUAUGUAUAAGCUAGU 14 distinguishing hsa-miR-664-002897UAUUCAUUUAUCCCCAGCCUACA 15 (Control + ILBD) hsa-miR-519a-4395526AAAGUGCAUCCUUUUAGAGUGU 16 vs PD hsa-miR-340#-002259UCCGUCUCAGUUACUUUAUAGC 17 hsa-miR-720-002895 UCUCGCUGGGGCCUCCA 18hsa-miR-142-3p-4373136 UGUAGUGUUUCCUACUUUAUGGA 19 hsa-miR-185-4395382UGGAGAGAAAGGCAGUUCCUGA 20 hsa-miR-213-000516 ACCAUCGACCGUUGAUUGUACC 21miRNA signature hsa-miR-664-002897 UAUUCAUUUAUCCCCAGCCUACA 22distinguishing ILBD hsa-miR-1285-002822 UCUGGGCAACAAAGUGAGACCU 23 vs PDhsa-miR-1183-002841 CACUGUAGGUGAUGGUGAGAGUGGGCA 24 hsa-miR-143-4395360UGAGAUGAAGCACUGUAGCUCA 25 hsa-miR-519a-4395526 AAAGUGCAUCCUUUUAGAGUGU 26hsa-miR-603-001566 CACACACUGCAAUUACUUUUGC 27 miRNA signaturehsa_miR_590_3P_002677 UAAUUUUAUGUAUAAGCUAGU 28 distinguishing PDhsa_miR_213_000516 ACCAUCGACCGUUGAUUGUACC 29 vs PDDnoADhsa_miR_409_3p_002332 GAAUGUUGCUCGGUGAACCCCU 30 hsa_miR_500_4395539UAAUCCUUGCUACCUGGGUGAGA 31 dme_miR_7_000268 UGGAAGACUAGUGAUUUUGUUGU 32hsa_miR_206_000510 UGGAAUGUAAGGAAGUGUGUGG 33 hsa_miR_629_001562GUUCUCCCAACGUAAGCCCAGC 34 All miRNA dme-miR-7-000268UGGAAGACUAGUGAUUUUGUUGU 35 differentially hsa-let-7c-4373167UGAGGUAGUAGGUUGUAUGGUU 36 expressed and/or hsa-let-7d-4395394AGAGGUAGUAGGUUGCAUAGU 37 correlated with hsa-let-7f-1#-002417CUAUACAAUCUAUUGCCUUCCC 38 clinical/pathological hsa-miR-100#-002142CAAGCUUGUAUCUAUAGGUAUG 39 findings hsa-miR-101#-002143UACAGUACUGUGAUAACUGAA 40 hsa-miR-101-4395364 CAGUUAUCACAGUGCUGAUGCU 41hsa-miR-105-4395278 UCAAAUGCUCAGACUCCUGUGGU 42 hsa-miR-106b#-002380CCGCACUGUGGGUACUUGCUGC 43 hsa-miR-107-4373154 AGCAGCAUUGUACAGGGCUAUCA 44hsa-miR-10b-4395329 UACCCUGUAGAACCGAAUUUGUG 45 hsa-miR-1183-002841CACUGUAGGUGAUGGUGAGAGUGGGCA 46 hsa-miR-1201-002781AGCCUGAUUAAACACAUGCUCUGA 47 hsa-miR-122-4395356 UGGAGUGUGACAAUGGUGUUUG48 hsa-miR-1224-3P-002752 CCCCACCUCCUCUCUCCUCAG 49 hsa-miR-1226#-002758GUGAGGGCAUGCAGGCCUGGAUGGGG 50 hsa-miR-1227-002769 CGUGCCACCCUUUUCCCCAG51 hsa-miR-1233-002768 UGAGCCCUGUCCUCCCGCAG 52 hsa-miR-1238-002927CUUCCUCGUCUGUCUGCCCC 53 hsa-miR-1244-002791 AAGUAGUUGGUUUGUAUGAGAUGGUU54 hsa-miR-124-4373295 UUAAGGCACGCGGUGAAUGCCA 55 hsa-miR-1248-002870ACCUUCUUGUAUAAGCACUGUGCUAAA 56 hsa-miR-1255B-002801CGGAUGAGCAAAGAAAGUGGUU 57 hsa-miR-1256-002850 AGGCAUUGACUUCUCACUAGCU 58hsa-miR-125a-5p-4395309 UCCCUGAGACCCUUUAACCUGUGA 59hsa-miR-125b2#-002158 UCACAAGUCAGGCUCUUGGGAC 60 hsa-miR-125b-4373148UCCCUGAGACCCUAACUUGUGA 61 hsa-miR-126#-000451 CAUUAUUACUUUUGGUACGCG 62hsa-miR-1260-002896 AUCCCACCUCUGCCACCA 63 hsa-miR-1264-002799CAAGUCUUAUUUGAGCACCUGUU 64 hsa-miR-1269-002789 CUGGACUGAGCCGUGCUACUGG 65hsa-miR-1270-002807 CUGGAGAUAUGGAAGAGCUGUGU 66 hsa-miR-1271-002779CUUGGCACCUAGCAAGCACUCA 67 hsa-miR-127-5p-4395340 CUGAAGCUCAGAGGGCUCUGAU68 hsa-miR-1282-002803 UCGUUUGCCUUUUUCUGCUU 69 hsa-miR-1290-002863UGGAUUUUUGGAUCAGGGA 70 hsa-miR-1291-002838 UGGCCCUGACUGAAGACCAGCAGU 71hsa-miR-129-3p-4373297 AAGCCCUUACCCCAAAAAGUAU 72 hsa-miR-1298-002861UUCAUUCGGCUGUCCAGAUGUA 73 hsa-miR-1300-002902 UUGAGAAGGAGGCUGCUG 74hsa-miR-1301-002827 UUGCAGCUGCCUGGGAGUGACUUC 75 hsa-miR-1303-002792UUUAGAGACGGGGUCUUGCUCU 76 hsa-miR-130a-4373145 CAGUGCAAUGUUAAAAGGGCAU 77hsa-miR-130b-4373144 CAGUGCAAUGAUGAAAGGGCAU 78 hsa-miR-132-4373143UAACAGUCUACAGCCAUGGUCG 79 hsa-miR-135a-4373140 UAUGGCUUUUUAUUCCUAUGUGA80 hsa-miR-135b#-002159 AUGUAGGGCUAAAAGCCAUGGG 81 hsa-miR-135b-4395372UAUGGCUUUUCAUUCCUAUGUGA 82 hsa-miR-136#-002100 CAUCAUCGUCUCAAAUGAGUCU 83hsa-miR-137-4373301 UAUUGCUUAAGAAUACGCGUAG 84 hsa-miR-138-2#-002144AGCUGGUGUUGUGAAUCAGGCCG 85 hsa-miR-138-4395395 GCUAUUUCACGACACCAGGGUU 86hsa-miR-141-4373137 UAACACUGUCUGGUAAAGAUGG 87 hsa-miR-142-3p-4373136UGUAGUGUUUCCUACUUUAUGGA 88 hsa-miR-143-4395360 UGAGAUGAAGCACUGUAGCUC 89hsa-miR-14395333 UGGAAUGUAAAGAAGUAUGUA 90 hsa-miR-144#-002148GGAUAUCAUCAUAUACUGUAAG 91 hsa-miR-144-002676 UACAGUAUAGAUGAUGUACU 92hsa-miR-145#-002149 GGAUUCCUGGAAAUACUGUUCU 93 hsa-miR-145-4395389GUCCAGUUUUCCCAGGAAUCCCU 94 hsa-miR-146a#-002163 CCUCUGAAAUUCAGUUCUUCAG95 hsa-miR-146b-5p-4373178 UGAGAACUGAAUUCCAUAGGCU 96hsa-miR-148a-4373130 UCAGUGCACUACAGAACUUUGU 97 hsa-miR-148b#-002160AAGUUCUGUUAUACACUCAGGC 98 hsa-miR-149#-002164 AGGGAGGGACGGGGGCUGUGC 99hsa-miR-151-5P-002642 UCGAGGAGCUCACAGUCUAGU 100 hsa-miR-153-4373305UUGCAUAGUCACAAAAGUGA 101 hsa-miR-154#-000478 AAUCAUACACGGUUGACCUAUU 102hsa-miR-15a-4373123 UAGCAGCACAUAAUGGUUUGUG 103 hsa-miR-181a2#-002317ACCACUGACCGUUGACUGUACC 104 hsa-miR-181a-4373117 AACAUUCAACGCUGUCGGUGAGU105 hsa-miR-183#-002270 UAUGGCACUGGUAGAAUUCACU 106 hsa-miR-183-4395380GUGAAUUACCGAAGGGCCAUAA 107 hsa-miR-184-4373113 UGGACGGAGAACUGAUAAGGGU108 hsa-miR-18a-4395533 UAAGGUGCAUCUAGUGCAGAUAG 109 hsa-miR-191#-002678GCUGCGCUUGGAUUUCGUCCCC 110 hsa-miR-192-4373108 CUGACCUAUGAAUUGACAGCC 111hsa-miR-193a-5p-4395392 UGGGUCUUUGCGGGCGAGAUGA 112 hsa-miR-195-4373105UAGCAGCACAGAAAUAUUGGC 113 hsa-miR-196b-4395326 UAGGUAGUUUCCUGUUGUUGGG114 hsa-miR-197-4373102 UUCACCACCUUCUCCACCCAGC 115 hsa-miR-198-4395384GGUCCAGAGGGGAGAUAGGUUC 116 hsa-miR-199a-5p-4373272CCCAGUGUUCAGACUACCUGUUC 117 hsa-miR-19b-4373098 UGUGCAAAUCCAUGCAAAACUGA118 hsa-miR-200b-4395362 UAAUACUGCCUGGUAAUGAUGA 119 hsa-miR-202-4395474AGAGGUAUAGGGCAUGGGAA 120 hsa-miR-203-4373095 GUGAAAUGUUUAGGACCACUAG 121hsa-miR-206-000510 UGGAAUGUAAGGAAGUGUGUGG 122 hsa-miR-211-4373088UUCCCUUUGUCAUCCUUCGCCU 123 hsa-miR-212-4373087 UAACAGUCUCCAGUCACGGCC 124hsa-miR-213-000516 ACCAUCGACCGUUGAUUGUACC 125 hsa-miR-21-4373090UAGCUUAUCAGACUGAUGUUGA 126 hsa-miR-217-4395448 UACUGCAUCAGGAACUGAUUGGA127 hsa-miR-218-4373081 UUGUGCUUGAUCUAACCAUGU 128hsa-miR-219-2-3p-4395501 AGAAUUGUGGCUGGACAUCUGU 129 hsa-miR-22#-002301AGUUCUUCAGUGGCAAGCUUUA 130 hsa-miR-220b-4395317 CCACCACCGUGUCUGACACUU131 hsa-miR-222#-002097 CUCAGUAGCCAGUGUAGAUCCU 132 hsa-miR-223-4395406UGUCAGUUUGUCAAAUACCCCA 133 hsa-miR-23a-4373074 AUCACAUUGCCAGGGAUUUCC 134hsa-miR-24-1#-002440 UGCCUACUGAGCUGAUAUCAGU 135 hsa-miR-25-4373071CAUUGCACUUGUCUCGGUCUGA 136 hsa-miR-26a-2#-002115 CCUAUUCUUGAUUACUUGUUUC137 hsa-miR-26b#-002444 CCUGUUCUCCAUUACUUGGCUC 138 hsa-miR-27a#-002445AGGGCUUAGCUGCUUGUGAGCA 139 hsa-miR-27b-4373068 UUCACAGUGGCUAAGUUCUGC 140hsa-miR-28-3p-4395557 CACUAGAUUGUGAGCUCCUGGA 141 hsa-miR-296-3p-4395212GAGGGUUGGGUGGAGGCUCUCC 142 hsa-miR-296-5p-4373066 AGGGCCCCCCCUCAAUCCUGU143 hsa-miR-29a#-002447 ACUGAUUUCUUUUGGUGUUCAG 144 hsa-miR-29a-4395223UAGCACCAUCUGAAAUCGGUUA 145 hsa-miR-29c-4395171 UAGCACCAUUUGAAAUCGGUUA146 hsa-miR-302a-4378070 UAAGUGCUUCCAUGUUUUGGUGA 147 hsa-miR-302d-000535UAAGUGCUUCCAUGUUUGAGUGU 148 hsa-miR-30a-3p-000416 CUUUCAGUCGGAUGUUUGCAGC149 hsa-miR-30b-4373290 UGUAAACAUCCUACACUCAGCU 150 hsa-miR-30c-1#-002108CUGGGAGAGGGUUGUUUACUCC 151 hsa-miR-30e-3p-000422 CUUUCAGUCGGAUGUUUACAGC152 hsa-miR-31#-002113 UGCUAUGCCAACAUAUUGCCAU 153 hsa-miR-31-4395390AGGCAAGAUGCUGGCAUAGCU 154 hsa-miR-320B-002844 AAAAGCUGGGUUGAGAGGGCAA 155hsa-miR-324-5p-4373052 CGCAUCCCCUAGGGCAUUGGUGU 156 hsa-miR-326-4373050CCUCUGGGCCCUUCCUCCAG 157 hsa-miR-328-4373049 CUGGCCCUCUCUGCCCUUCCGU 158hsa-miR-330-5p-4395341 UCUCUGGGCCUGUGUCUUAGGC 159 hsa-miR-337-3p-002157CUCCUAUAUGAUGCCUUUCUUC 160 hsa-miR-338-3p-4395363 UCCAGCAUCAGUGAUUUUGUUG161 hsa-miR-339-5p-4395368 UCCCUGUCCUCCAGGAGCUCACG 162hsa-miR-33b-4395196 GUGCAUUGCUGUUGCAUUGC 163 hsa-miR-340#-002259UCCGUCUCAGUUACUUUAUAGC 164 hsa-miR-340-4395369 UUAUAAAGCAAUGAGACUGAUU165 hsa-miR-345-4395297 GCUGACUCCUAGUCCAGGGCUC 166 hsa-miR-34a#-002316CAAUCAGCAAGUAUACUGCCCU 167 hsa-miR-34a-4395168 UGGCAGUGUCUUAGCUGGUUGU168 hsa-miR-34b-000427 UAGGCAGUGUCAUUAGCUGAUUG 169hsa-miR-34c-5p-4373036 AGGCAGUGUAGUUAGCUGAUUGC 170hsa-miR-362-5p-4378092 AAUCCUUGGAACCUAGGUGUGAGU 171 hsa-miR-363#-001283CGGGUGGAUCACGAUGCAAUUU 172 hsa-miR-363-4378090 AAUUGCACGGUAUCCAUCUGUA173 hsa-miR-365-4373194 UAAUGCCCCUAAAAAUCCUUAU 174 hsa-miR-367-4373034AAUUGCACUUUAGCAAUGGUGA 175 hsa-miR-373-4378073 GAAGUGCUUCGAUUUUGGGGUGU176 hsa-miR-374b#-002391 CUUAGCAGGUUGUAUUAUCAUU 177 hsa-miR-374b-4381045AUAUAAUACAACCUGCUAAGUG 178 hsa-miR-376c-4395233 AACAUAGAGGAAAUUCCACGU179 hsa-miR-378-000567 CUCCUGACUCCAGGUCCUGUGU 180 hsa-miR-380-5p-000570UGGUUGACCAUAGAACAUGCGC 181 hsa-miR-381-4373020 UAUACAAGGGCAAGCUCUCUGU182 hsa-miR-382-4373019 GAAGUUGUUCGUGGUGGAUUCG 183 hsa-miR-383-4373018AGAUCAGAAGGUGAUUGUGGCU 184 hsa-miR-409-3p-002332 GAAUGUUGCUCGGUGAACCCCU185 hsa-miR-411-4381013 UAGUAGACCGUAUAGCGUACG 186 hsa-miR-424#-002309CAAAACGUGAGGCGCUGCUAU 187 hsa-miR-424-4373201 CAGCAGCAAUUCAUGUUUUGAA 188hsa-miR-431-4395173 UGUCUUGCAGGCCGUCAUGCA 189 hsa-miR-432-001026UCUUGGAGUAGGUCAUUGGGUGG 190 hsa-miR-448-4373206 UUGCAUAUGUAGGAUGUCCCAU191 hsa-miR-449b-4381011 AGGCAGUGUAUUGUUAGCUGGC 192 hsa-miR-450a-4395414UUUUGCGAUGUGUUCCUAAUAU 193 hsa-miR-454-4395434 UAGUGCAAUAUUGCUUAUAGGGU194 hsa-miR-483-3p-002339 UCACUCCUCUCCUCCCGUCUU 195 hsa-miR-484-4381032UCAGGCUCAGUCCCCUCCCGAU 196 hsa-miR-486-5p-4378096 UCCUGUACUGAGCUGCCCCGAG197 hsa-miR-488-4395468 UUGAAAGGCUAUUUCUUGGUC 198 hsa-miR-489-4395469GUGACAUCACAUAUACGGCAGC 199 hsa-miR-497-001043 CAGCAGCACACUGUGGUUUGU 200hsa-miR-499-5p-4381047 UUAAGACUUGCAGUGAUGUUUAA 201 hsa-miR-500-001046AUGCACCUGGGCAAGGAUUCUG 202 hsa-miR-511-4373236 GUGUCUUUUGCUCUGCAGUCA 203hsa-miR-516-3p-001149 UGCUUCCUUUCAGAGGGU 204 hsa-miR-516b-4395172AUCUGGAGGUAAGAAGCACUUU 205 hsa-miR-517#-001113 CCUCUAGAUGGAAGCACUGUCU206 hsa-miR-518a-3p-4395508 GAAAGCGCUUCCCUUUGCUGGA 207hsa-miR-518b-4373246 CAAAGCGCUCCCCUUUAGAGGU 208 hsa-miR-519a-4395526AAAGUGCAUCCUUUUAGAGUGU 209 hsa-miR-520c-3p-002400 AAAGUGCUUCCUUUUAGAGGGU210 hsa-miR-520D-3P-002743 AAAGUGCUUCUCUUUGGUGGGU 211hsa-miR-520g-4373257 ACAAAGUGCUUCCCUUUAGAGUGU 212 hsa-miR-521-4373259AACGCACUUCCCUUUAGAGUGU 213 hsa-miR-539-4378103 GGAGAAAUUAUCCUUGGUGUGU214 hsa-miR-541-4395312 UGGUGGGCACAGAAUCUGGACU 215 hsa-miR-543-002376AAACAUUCGCGGUGCACUUCUU 216 hsa-miR-545-4395378 UCAGCAAACAUUUAUUGUGUGC217 hsa-miR-548b-5p-4395519 AAAAGUAAUUGUGGUUUUGGCC 218hsa-miR-548c-5p-4395540 AAAAGUAAUUGCGGUUUUUGCC 219 hsa-miR-5481-002909AAAAGUAAUUGCGGAUUUUGCC 220 hsa-miR-551b#-002346 GAAAUCAAGCGUGGGUGAGACC221 hsa-miR-552-001520 AACAGGUGACUGGUUAGACAA 222 hsa-miR-559-001527UAAAGUAAAUAUGCACCAAAA 223 hsa-miR-566-001533 GGGCGCCUGUGAUCCCAAC 224hsa-miR-571-001613 UGAGUUGGCCAUCUGAGUGAG 225 hsa-miR-574-3p-4395460CACGCUCAUGCACACACCCACA 226 hsa-miR-577-002675 UAGAUAAAAUAUUGGUACCUG 227hsa-miR-582-3p-4395510 UAACUGGUUGAACAACUGAACC 228 hsa-miR-589-001543UCAGAACAAAUGCCGGUUCCCAGA 229 hsa-miR-590-3P-002677 UAAUUUUAUGUAUAAGCUAGU230 hsa-miR-590-5p-4395176 GAGCUUAUUCAUAAAAGUGCAG 231 hsa-miR-592-001546UUGUGUCAAUAUGCGAUGAUGU 232 hsa-miR-593-001547 AGGCACCAGCCAGGCAUUGCUCAGC233 hsa-miR-597-4380960 UGUGUCACUCGAUGACCACUGU 234 hsa-miR-601-001558UGGUCUAGGAUUGUUGGAGGAG 235 hsa-miR-604-001567 AGGCUGCGGAAUUCAGGAC 236hsa-miR-608-001571 AGGGGUGGUGUUGGGACAGCUCCGU 237 hsa-miR-616-4395525AGUCAUUGGAGGGUUUGAGCAG 238 hsa-miR-617-001591 AGACUUCCCAUUUGAAGGUGGC 239hsa-miR-618-4380996 AAACUCUACUUGUCCUUCUGAGU 240 hsa-miR-622-001553ACAGUCUGCUGAGGUUGGAGC 241 hsa-miR-625#-002432 GACUAUAGAACUUUCCCCCUCA 242hsa-miR-626-001559 AGCUGUCUGAAAAUGUCUU 243 hsa-miR-628-3p-002434UCUAGUAAGAGUGGCAGUCGA 244 hsa-miR-628-5p-4395544 AUGCUGACAUAUUUACUAGAGG245 hsa-miR-629-001562 UGGGUUUACGUUGGGAGAACU 246 hsa-miR-630-001563AGUAUUCUGUACCAGGGAAGGU 247 hsa-miR-635-001578 ACUUGGGCACUGAAACAAUGUCC248 hsa-miR-638-001582 AGGGAUCGCGGGCGGGUGGCGGCCU 249 hsa-miR-639-001583AUCGCUGCGGUUGCGAGCGCUGU 250 hsa-miR-641-001585 AAAGACAUAGGAUAGAGUCACCUC251 hsa-miR-644-001596 AGUGUGGCUUUCUUAGAGC 252 hsa-miR-649-001602AAACCUGUGUUGUUCAAGAGUC 253 hsa-miR-652-4395463 AAUGGCGCCACUAGGGUUGUG 254hsa-miR-654-3p-4395350 UAUGUCUGCUGACCAUCACCUU 255 hsa-miR-659-001514CUUGGUUCAGGGAGGGUCCCCA 256 hsa-miR-660-4380925 UACCCAUUGCAUAUCGGAGUUG257 hsa-miR-661-001606 UGCCUGGGUCUCUGGCCUGCGCGU 258 hsa-miR-663B-002857GGUGGCCCGGCCGUGCCUGAGG 259 hsa-miR-664-002897 UAUUCAUUUAUCCCCAGCCUACA260 hsa-miR-671-3p-4395433 UCCGGUUCUCAGGGCUCCACC 261 hsa-miR-708-4395452AAGGAGCUUACAAUCUAGCUGGG 262 hsa-miR-720-002895 UCUCGCUGGGGCCUCCA 263hsa-miR-758-4395180 UUUGUGACCUGGUCCACUAACC 264 hsa-miR-767-5p-001993UGCACCAUGGUUGUCUGAGCAUG 265 hsa-miR-769-5p-001998 UGAGACCUCUGGGUUCUGAGCU266 hsa-miR-873-4395467 GCAGGAACUUGUGAGUCUCCU 267 hsa-miR-876-3p-4395336UGGUGGUUUACAAAGUAAUUCA 268 hsa-miR-885-5p-4395407 UCCAUUACACUACCCUGCCUCU269 hsa-miR-886-3p-4395305 CGCGGGUGCUUACUGACCCUU 270hsa-miR-886-5p-4395304 CGGGUCGGAGUUAGCUCAAGCGG 271 hsa-miR-9#-002231AUAAAGCUAGAUAACCGAAAGU 272 hsa-miR-920-002150 GGGGAGCUGUGGAAGCAGUA 273hsa-miR-922-002152 GCAGCAGAGAAUAGGACUACGUC 274 hsa-miR-92a-1#-002137AGGUUGGGAUCGGUUGCAAUGCU 275 hsa-miR-92a-4395169 UAUUGCACUUGUCCCGGCCUGU276 hsa-miR-9-4373285 UCUUUGGUUAUCUAGCUGUAUGA 277 hsa-miR-98-4373009UGAGGUAGUAAGUUGUAUUGUU 278 hsa-miR-99a#-002141 CAAGCUCGCUUCUAUGGGUCUG279 hsa-miR-99b-4373007 CACCCGUAGAACCGACCUUGCG 280 mmu-let-7d#-001178CUAUACGACCUGCUGCCUUUCU 281 rno-miR-29c#-001818 UGACCGAUUUCUCCUGGUGUUC282 rno-miR-7#-001338 CAACAAAUCACAGUCUGCCAUA 283

As shown in the Examples discovered are miRNAs that are useful asdiagnostic markers and therapeutic targets for the treatment andprevention of Parkinson's Disease and related disorders. As used herein,disorders related to Parkinson's Disease may include, but are notlimited to, Alzheimer's, dementia, cognitive impairment, pre-symptomaticParkinson's Disease, and Lewis Body disease.

miRNA or microRNA refer to 19-25 nucleotide (nt) non-coding RNAs derivedfrom endogenous genes that act as post-transcriptional regulators ofgene expression. They are processed from longer (ca 70-80 nt)hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme Dicer.miRNAs assemble in ribonucleoprotein complexes termed miRNPs andrecognise their target sites by antisense complementarity therebymediating down-regulation of their target genes. Near-perfect or perfectcomplementarity between the miRNA and its target site results in targetmRNA cleavage, whereas limited complementarity between the miRNA and thetarget site results in translational inhibition of the target gene.

Compositions

In certain aspects, provided are pharmaceutical compositions. Thepharmaceutical compositions may include an oligonucleotide thatdown-regulates the over-expression of at least one miRNA of SEQ ID NOs:1-283. The oligonucleotide may be complementary to the nucleotidesequence of at least one of SEQ ID NOs: 1-283. The oligonucleotide mayhybridize under stringent conditions to a nucleotide sequence of atleast one of SEQ ID NOs: 1-283. The oligonucleotide may comprise anynucleic acid. “Nucleic acid” includes double- and single-stranded DNA ofdifferent lengths, as well as double- and single stranded RNA ofdifferent lengths, or synthetic variants thereof. Thus, the nucleic acidmay be, for example, DNA, cDNA, RNA, sRNA, locked nucleotide acid (LNA),HNA, peptide nucleic acid (PNA), or any other variant hereof.

As used herein, “complementary” or “substantially complementary” refersto the hybridization or base pairing between nucleotides or nucleicacids, such as, for instance, between the two strands of a doublestranded DNA molecule or between an oligonucleotide primer and a primerbinding site on a single stranded nucleic acid to be sequenced oramplified. Complementary nucleotides are, generally, A and T (or A andU), or C and G. Two single stranded RNA or DNA molecules are said to besubstantially complementary when the nucleotides of one strand,optimally aligned and with appropriate nucleotide insertions ordeletions, pair with at least about 80% of the nucleotides of the otherstrand, usually at least about 90% to 95%, and more preferably fromabout 98 to 100%

The oligonucleotide may be at least about 10 nucleotides (nt), at leastabout 11 nt, at least about 12 nt, at least about 13 nt, at least about14 nt, at least about 15 nt, at least about 16 nt, at least about 17 nt,at least about 18 nt, at least about 19 nt, or at least about 20 nt inlength. The oligonucleotide may be less than about 100 nt, less thanabout 90 nt, less than about 80 nt, less than about 70 nt, less thanabout 60 nt, or less than about 50 nt in length. This may include rangesof about 10 nt to about 50 nt, about 12 nt to about 40 nt, or about 15nt to about 30 nt in length.

The oligonucleotide may be capable of reducing expression of the miRNAby at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, or at least about 60%. Theoligonucleotide may be capable of reducing expression of the miRNA byless than about 99%, less than about 98%, less than about 95%, less thanabout 90%, less than about 85%, less than about 80%, or less than about75%. This may include ranges of about 10% to about 99%, about 20% toabout 98%, about 30% to about 90%, or about 40% to about 80%.

The oligonucleotide may be chemically modified. Chemical modificationsmay include addition of an imaging agent or reporter molecule. As usedherein, an “imaging agent” or “reporter molecule” is any entity whichenhances visualization or detection of the oligonucleotide. Any type ofdetectable reporter molecule/imaging agent can be used in the methodsdisclosed herein. Such detectable molecules are known in the art andinclude, for example, magnetic beads, fluorophores, radionuclides,nuclear stains (e.g., DAPI). For example, an imaging agent can include acompound that comprises an unstable isotope (i.e., a radionuclide) or afluorescent moiety, such as Cy-5, Alexa 647, Alexa 555, Alexa 488,fluorescein, rhodamine, and the like. Suitable radionuclides includeboth alpha- and beta-emitters. In some embodiments, the targetingvehicle is labeled. In other embodiments, suitable radioactive moietiesinclude labeled polynucleotides and polypeptides which can be coupled tothe targeting vehicle.

In some embodiments, the imaging agent comprises a radionuclide such as,for example, a radionuclide that emits low-energy electrons (e.g., thosethat emit photons with energies as low as 20 keV). Such nuclides canirradiate the cell to which they are delivered without irradiatingsurrounding cells or tissues. Non-limiting examples of radionuclidesthat are can be delivered to cells include ¹³⁷Cs, ¹⁰³Pd, ¹¹¹In, ¹²⁵I,²¹¹At, ²¹²Bi and ²¹³Bi, among others known in the art. Further imagingagents suitable for delivery to a cell in accordance with someembodiments include paramagnetic species for use in MRI imaging,echogenic entities for use in ultrasound imaging, fluorescent entitiesfor use in fluorescence imaging (including quantum dots), andlight-active entities for use in optical imaging. A suitable species forMRI imaging is a gadolinium complex of diethylenetriamine pentaceticacid (DTPA). For positron emission tomography (PET), ¹⁸F or ¹¹C may bedelivered.

Methods

In further aspects, the disclosure relates to methods of diagnosingParkinson's Disease (PD) in a subject. The methods may include detectingthe level of expression of at least one miRNA of SEQ ID NOs: 1-283 in abiological sample from the subject, and comparing the level ofexpression in the sample to the level of expression in a reference. Anincreased or decreased level of expression in the sample compared to thelevel of expression in the reference may identify the subject as havingPD or who is at risk of developing PD.

Various detection methods that can be employed for the methods describedherein, and the present invention includes all the mentioned methods,but is not limited to any of these. Detection methods may include use ofa probe. A probe is a specific sequence of a nucleic acid used to detectnucleic acids by hybridization. A probe may be labelled, tagged orimmobilized or otherwise modified according to the requirements of thedetection method chosen. A label or a tag is an entity making itpossible to identify a compound to which it is associated. It is withinthe scope of the present invention to employ probes that are labelled ortagged by any means known in the art such as but not limited to:radioactive labelling, fluorescent labelling and enzymatic labelling.Furthermore the probe, labeled or not, may be immobilized to facilitatedetection according to the detection method of choice and this may beaccomplished according to the preferred method of the particulardetection method. Detection methods may also include in situhybridization, PCR, microarrays, Northern blot analysis, and affinitymatrices.

In some embodiments, RT-PCR may be used as the detection method. In someembodiments, the detection method may include quantitative real-timeRT-PCR, for example, the TaqMan® MicroRNA assay (Applied Biosystems) maybe used for detection of expression of the miRNAs described herein.

In some embodiments, a kit may be provided with reagents to measure atleast one of the miRNAs. In some embodiments, a kit may be provided tomeasure a miRNA signature. By way of non-limiting example, the kit mayinclude reagents to measure a miRNA signature where the miRNA signatureincludes at least one of SEQ ID NOs: 204, 110, 192, 200, 244, 21, 134,144, 198, 234, 143, 261, 255, and 179 (miR-516-3p, miR-191#, miR-449b,miR-497, miR-628-3p, miR-213, miR-23a, miR-29a#, miR-488, miR-597,miR-296-5p, miR-671-3p, miR-654-3p, and miR-99a#) or a combinationthereof and in some embodiments includes all 14. An embodiment of thekit may include reagents to measure a miRNA signature where the miRNAsignature includes at least one of SEQ ID NOs: 1-13, (miR-590-3p,miR-213, miR-9#, miR-191#, miR-497, miR-664, miR-99a#, miR-1183,miR-340#, miR-628-3p, miR-7#, miR-29a#, miR-142-3p) or combinationsthereof and in some embodiments includes all 13. An embodiment of thekit may include reagents to measure a miRNA signature where the miRNAsignature includes at least one of SEQ ID NOs: 14-21 (miR-590-3p,miR-664, miR-519a, miR-340#, miR-720, miR-142-3p, miR-185, and miR-213)or combinations thereof an in some embodiments includes all 8. Anembodiment of the kit may include reagents to measure a miRNA signaturewhere the miRNA signature includes at least one of SEQ ID NOs: 22-27(miR-664, miR-1285, miR-1183, miR-143, miR-519a, miR-603) orcombinations thereof an in some embodiments includes all 6. Anembodiment of the kit may include reagents to measure a miRNA signaturewhere the miRNA signature includes at least one of SEQ ID NOs: 28-34(miR-590-3p, miR-213, miR-409-3p, miR-500, miR-7, miR-206, and miR-629)or combinations thereof an in some embodiments includes all 7.Additional combinations for the kit for measuring a miRNA signature arealso possible.

In some embodiments, the expression level of one or more miRNAs of SEQID NOS 1-283 may be compared to a reference expression level usingstatistical analysis with a computer to implement the statisticalanalysis. In some embodiments Support Vector Machine Recursive FeatureSelection (SVM-RFE) and SVM classification analysis from JMP Genomics(SAS Institute) is used to compare the miRNA expression levels. In someembodiments, the data for the array assays will be first analyzed usingRQ manager 1.2 software (Applied Biosystems) to obtain raw expressionlevels of all the miRNAs in each sample, represented by C^(T). In someembodiments, the miRNA expression profiles of ILBD, PD, and PDDnoADpatients may be compared to age-matched normal controls in the CSF orSNs. The miRNA profiles of ILBD, PD, and PDDnoAD patients may becompared to each other. Changes in the levels of miRNA expression ineach pair will be evaluated by the DDCt or Δ(ΔC^(T)). In someembodiments, StatMiner software (Integromics) may be used forbioinformatic and statistical analysis.

The subject may be any mammal. Mammals include including humans, mice,rats, rabbits, cats, and dogs. The subject is preferably human. Thesubject may be predisposed for a disease or suffering from a disease.

A sample may be a small part of a subject, representative of the whole,and may be constituted by a biopsy or a body fluid sample. Biopsies aresmall pieces of tissue and may be fresh, frozen or fixed, such asformalin-fixed and paraffin embedded (FFPE). Body fluid samples may beblood, plasma, serum, urine, sputum, cerebrospinal fluid, milk, orductal fluid samples and may likewise be fresh, frozen or fixed.Suitably, the sample comprises blood. Samples may be removed surgically,by extraction, i.e. by hypodermic or other types of needles, bymicrodissection, or laser capture.

The reference may be any suitable control sample known in the art, suchas, for example, a sample from a normal, healthy subject. The referencemay be a sample from the same subject prior to demonstration of diseasesymptoms or prior to diagnosis with Parkinson's Disease. The referencemay be a “standardized” sample, such as a sample comprising material ordata from several samples, preferably also from several individuals. Astandardized sample may comprise either normal or diseased samplematerial or data.

Before analyzing the sample, it may be desirable to perform one or moresample preparation operations upon the sample. Typically, these samplepreparation operations will include such manipulations as concentration,suspension, extraction of intracellular material, e.g., nucleic acidsfrom tissue/whole cell samples and the like, amplification of nucleicacids, fragmentation, transcription, labeling and/or extensionreactions. Nucleic acids, especially RNA and specifically miRNA can beisolated using any techniques known in the art. There are two mainmethods for isolating RNA: phenol-based extraction and silica matrix orglass fiber filter (GFF)-based binding. Phenol-based reagents contain acombination of denaturants and RNase inhibitors for cell and tissuedisruption and subsequent separation of RNA from contaminants.Phenol-based isolation procedures can recover RNA species in the10-200-nucleotide range e.g., miRNAs, 5S rRNA, 5.8S rRNA, and U1 snRNA.If a sample of “total” RNA was purified by the popular silica matrixcolumn or GFF procedure, it may be depleted in small RNAs. Extractionprocedures such as those using Trizol or TriReagent, however will purifyall RNAs, large and small, and are the recommended methods for isolatingtotal RNA from biological samples that will contain miRNAs/siRNAs. Anymethod required for the processing of a sample prior to detection by anyof the herein mentioned methods falls within the scope of the presentinvention. These methods are typically well known by a person skilled inthe art.

Other aspects of the disclosure provide methods for treating,preventing, or reducing the risk of PD associated with aberrantexpression of a miRNA in a cell, tissue, or animal. The methods mayinclude contacting the cell, tissue, or animal with a pharmaceuticalcomposition as detailed above. Aberrant expression includesup-regulation and down-regulation of the miRNA. In some embodiments,miRNA is up-regulated.

“Administration” or “administering,” as used herein, refers toproviding, contacting, and/or delivery of a compound or compounds by anyappropriate route to achieve the desired effect. Administration mayinclude, but is not limited to, oral, sublingual, parenteral (e.g.,intravenous, subcutaneous, intracutaneous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional or intracranial injection), transdermal, topical, buccal,rectal, vaginal, nasal, ophthalmic, via inhalation, and implants.

“Contacting,” as used herein as in “contacting a cell,” refers tocontacting a cell directly or indirectly in vitro, ex vivo, or in vivo(i.e. within a subject, such as a mammal). Contacting a cell, which alsoincludes “reacting” a cell, can occur as a result of administration to asubject. Contacting encompasses administration to a cell, tissue,mammal, subject, patient, or human. Further, contacting a cell includesadding an agent to a cell culture. Other suitable methods may includeintroducing or administering an agent to a cell, tissue, mammal,subject, or patient using appropriate procedures and routes ofadministration as defined herein.

As used herein, the term “treat” or “treating” a subject having adisorder refers to administering a regimen to the subject, such that atleast one symptom of the disorder is cured, healed, alleviated,relieved, altered, remedied, ameliorated, or improved. Treating includesadministering an amount effective to alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disorder or the symptoms of thedisorder. The treatment may inhibit deterioration or worsening of asymptom of a disorder.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including but not limited to”) unless otherwise noted.All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to illustrate aspects andembodiments of the disclosure and does not limit the scope of theclaims.

EXAMPLES Example 1 Putamen miRNAs are Involved in the Pathogenesis of PDand are Novel Therapeutic Targets for Treatment of PD

Among the differentially expressed miRNAs in the putamen, we identified51 miRNAs that are dysregulated among ILBD, PD and PDD, and normalcontrols (Tables 3-9; FIG. 1); 59 miRNAs that are dysregulated in Stages2, 3, and 4 of Lewy Body diseases when compared to normal controls(Tables 10-16; FIG. 2).

TABLE 3 miRNAs Differentially Expressed in Putamen among DifferentDiagnostic Groups (p < 0.05; at least 2 fold change) (51 in total)Diagnosis Control ILBD PD PDDnoAD Control 27 (11, 16) 16 (7, 9) 11 (3,8) ILBD 27 (11, 16) 14 (8, 6) 18 (10, 8) PD 16 (7, 9) 14 (8, 6) 12 (6,6) PDDnoAD 11 (3, 8) 18 (10, 8) 12 (6, 6) *Numbers in parentheses arenumbers of (upregulated, downregulated) miRNAs.

TABLE 4 miRNAs differentially expressed in ILBD compared to normalcontrols (corrected p value < 0.05, fold change > 2 folds) miRNAsp-value Fold change (ILBD/Ctl) hsa-miR-1224-3P-002752 0.007 10.69hsa-miR-767-5p-001993 0.008 5.31 hsa-miR-431-4395173 0.009 4.25mmu-let-7d#-001178 0.013 3.46 hsa-miR-183-4395380 0.000 3.37hsa-miR-873-4395467 0.015 2.68 hsa-miR-33b-4395196 0.040 2.55hsa-miR-516b-4395172 0.015 2.53 hsa-miR-127-5p-4395340 0.030 2.32hsa-miR-489-4395469 0.001 2.08 hsa-miR-1270-002807 0.029 2.06hsa-miR-34b-000427 0.008 0.20 hsa-miR-26a-2#-002115 0.002 0.22hsa-miR-638-001582 0.000 0.22 hsa-miR-1290-002863 0.002 0.23hsa-miR-302d-000535 0.016 0.23 hsa-miR-1248-002870 0.020 0.24hsa-miR-922-002152 0.024 0.26 hsa-miR-30c-1#-002108 0.000 0.30hsa-miR-92a-1#-002137 0.002 0.31 hsa-miR-497-001043 0.000 0.34hsa-miR-1291-002838 0.005 0.36 hsa-miR-1244-002791 0.009 0.40hsa-miR-635-001578 0.009 0.43 hsa-miR-601-001558 0.009 0.44hsa-miR-1226#-002758 0.043 0.47 hsa-miR-106b#-002380 0.002 0.47

TABLE 5 miRNAs differentially expressed in PD compared to normalcontrols (corrected p value < 0.05, fold change > 2 folds) miRNAsp-value Fold change (PD/Ctl) hsa-miR-566-001533 0.01 15.96hsa-miR-767-5p-001993 0.01 7.39 hsa-miR-1-4395333 0.01 2.94hsa-miR-33b-4395196 0.03 2.88 hsa-miR-217-4395448 0.01 2.17hsa-miR-424-4373201 0.04 2.17 hsa-miR-876-3p-4395336 0.02 2.12hsa-miR-516-3p-001149 0.05 0.11 hsa-miR-644-001596 0.02 0.15dme-miR-7-000268 0.05 0.27 hsa-miR-30c-1#-002108 0.01 0.38hsa-miR-720-002895 0.04 0.39 hsa-miR-106b#-002380 0.00 0.42hsa-miR-551b#-002346 0.02 0.46 hsa-miR-125b-2#-002158 0.02 0.48hsa-miR-1256-002850 0.01 0.48

TABLE 6 miRNAs differentially expressed in PDD compared to normalcontrols (corrected p value < 0.05, fold change > 2 folds) miRNAsp-value Fold change (PDD/Ctl) hsa-miR-518a-3p-4395508 0.011 2.910hsa-miR-520g-4373257 0.045 2.891 hsa-miR-337-3p-002157 0.015 2.851hsa-miR-516-3p-001149 0.042 0.090 hsa-miR-644-001596 0.042 0.183hsa-miR-922-002152 0.013 0.286 hsa-miR-193a-3p-4395361 0.017 0.300hsa-miR-92a-1#-002137 0.038 0.371 hsa-miR-30c-1#-002108 0.004 0.373hsa-miR-1291-002838 0.017 0.415 hsa-miR-15a-4373123 0.039 0.472

TABLE 7 miRNAs differentially expressed in PD compared to ILBD(corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Foldchange (PD/ILBD) hsa-miR-302d-000535 0.004 6.208 hsa-miR-23a-43730740.037 4.953 hsa-miR-1290-002863 0.018 3.974 hsa-miR-26a-2#-002115 0.0253.090 hsa-miR-638-001582 0.005 3.028 hsa-miR-886-3p-4395305 0.049 2.740hsa-miR-548c-5p-4395540 0.040 2.241 hsa-miR-92a-1#-002137 0.010 2.141hsa-miR-1224-3P-002752 0.049 0.273 hsa-miR-122-4395356 0.019 0.308hsa-miR-302a-4378070 0.009 0.357 hsa-miR-424#-002309 0.032 0.380hsa-miR-486-5p-4378096 0.010 0.409 hsa-miR-541-4395312 0.004 0.455

TABLE 8 miRNAs differentially expressed in PDD compared to ILBD(corrected p value < 0.05, fold change > 2 folds) miRNAs p-value foldchange (PDD/ILBD) hsa-miR-302d-000535 0.008 5.830 hsa-miR-26a-2#-0021150.002 4.415 hsa-miR-630-001563 0.046 3.232 hsa-miR-659-001514 0.0303.229 hsa-miR-641-001585 0.006 2.826 hsa-miR-638-001582 0.011 2.586hsa-miR-125b-2#-002158 0.044 2.458 hsa-miR-1255B-002801 0.010 2.387hsa-miR-337-3p-002157 0.048 2.190 hsa-miR-380-5p-000570 0.009 2.058hsa-miR-202-4395474 0.005 0.260 hsa-miR-873-4395467 0.001 0.279hsa-miR-767-5p-001993 0.040 0.298 hsa-miR-431-4395173 0.010 0.317hsa-miR-1224-3P-002752 0.050 0.371 hsa-miR-24-1#-002440 0.034 0.408hsa-miR-15a-4373123 0.002 0.455 hsa-miR-339-5p-4395368 0.001 0.494

TABLE 9 miRNAs differentially expressed in PDD compared to PD (correctedp value < 0.05, fold change > 2 folds) miRNAs p-value Fold Change(PDD/PD) dme-miR-7-000268 0.031 4.086 hsa-miR-135b#-002159 0.049 2.608hsa-miR-641-001585 0.003 2.553 hsa-miR-149#-002164 0.006 2.490hsa-miR-154#-000478 0.007 2.291 hsa-miR-521-4373259 0.028 2.192hsa-miR-566-001533 0.035 0.115 hsa-miR-767-5p-001993 0.025 0.214hsa-miR-202-4395474 0.008 0.321 hsa-miR-548b-5p-4395519 0.025 0.356hsa-miR-217-4395448 0.011 0.414 hsa-miR-326-4373050 0.042 0.463

TABLE 10 miRNAs Differentially Expressed in Putamen among DifferentStage Groups by the Unified Staging System for LBD (p < 0.05; at least 2fold change). (59 in total) Stages 0 2 3 4 0 29 (9, 20) 24 (11, 13) 10(3/7) 2 29 (9, 20) 9 (4, 5) 13 (7, 6) 3 24 (11, 13) 9 (4, 5) 7 (3, 4) 410 (3/7) 13 (7, 6) 7 (3, 4) *Numbers in parentheses are numbers of(upregulated, downregulated) miRNAs.

TABLE 11 miRNAs differentially expressed in the putamen of Stage IIcompared to controls (corrected p value < 0.05, fold change > 2 folds)miRNAs p-value Fold change (Stage2/0) hsa-miR-1224-3P-002752 0.016 8.494hsa-miR-767-5p-001993 0.027 5.601 mmu-let-7d#-001178 0.008 3.631hsa-miR-33b-4395196 0.035 2.762 hsa-miR-516b-4395172 0.033 2.435hsa-miR-183-4395380 0.006 2.427 hsa-miR-1-4395333 0.031 2.393hsa-miR-127-5p-4395340 0.048 2.089 hsa-miR-489-4395469 0.001 2.031hsa-miR-644-001596 0.015 0.157 hsa-miR-1290-002863 0.003 0.250hsa-miR-1248-002870 0.015 0.254 hsa-miR-638-001582 0.001 0.269hsa-miR-639-001583 0.017 0.274 hsa-miR-1303-002792 0.025 0.284hsa-miR-30c-1#-002108 0.000 0.310 hsa-miR-378-000567 0.041 0.318hsa-miR-26a-2#-002115 0.036 0.326 hsa-miR-1291-002838 0.002 0.328hsa-miR-616-4395525 0.015 0.342 hsa-miR-922-002152 0.028 0.346hsa-miR-1244-002791 0.000 0.358 hsa-miR-548c-5p-4395540 0.037 0.366hsa-miR-92a-1#-002137 0.015 0.385 hsa-miR-497-001043 0.000 0.392hsa-miR-601-001558 0.012 0.399 hsa-miR-720-002895 0.046 0.430hsa-miR-1260-002896 0.004 0.452 hsa-miR-635-001578 0.041 0.495

TABLE 12 miRNAs differentially expressed in the putamen of Stage IIIcompared to controls (corrected p value < 0.05, fold change > 2 folds)miRNAs p-value Fold change (Stage 3/0) hsa-miR-566-001533 0.037 13.485hsa-miR-1224-3P-002752 0.016 10.196 hsa-miR-98-4373009 0.046 4.805hsa-miR-767-5p-001993 0.006 3.802 hsa-miR-1-4395333 0.039 3.115hsa-miR-516b-4395172 0.018 2.728 hsa-miR-33b-4395196 0.049 2.448hsa-miR-183-4395380 0.017 2.382 hsa-miR-424-4373201 0.017 2.288hsa-miR-127-5p-4395340 0.033 2.144 hsa-miR-1270-002807 0.049 2.080hsa-miR-34b-000427 0.031 0.231 hsa-miR-922-002152 0.037 0.286hsa-miR-30c-1#-002108 0.001 0.323 hsa-miR-106b#-002380 0.000 0.353hsa-miR-199a-5p-4373272 0.047 0.357 hsa-miR-520D-3P-002743 0.005 0.370hsa-miR-100#-002142 0.000 0.400 hsa-miR-638-001582 0.022 0.401hsa-miR-1226#-002758 0.023 0.408 hsa-miR-92a-1#-002137 0.039 0.411hsa-miR-374b#-002391 0.005 0.436 hsa-miR-497-001043 0.006 0.460hsa-miR-1260-002896 0.009 0.463

TABLE 13 miRNAs differentially expressed in the putamen of Stage IVcompared to controls (corrected p value < 0.05, fold change > 2 folds)miRNAs p-value Fold change (Stage 4/0) hsa-miR-518a-3p-4395508 0.0272.605 hsa-miR-1269-002789 0.043 2.424 hsa-miR-876-3p-4395336 0.003 2.310hsa-miR-516-3p-001149 0.025 0.095 hsa-miR-644-001596 0.038 0.192hsa-miR-639-001583 0.022 0.257 hsa-miR-193a-3p-4395361 0.029 0.286hsa-miR-30c-1#-002108 0.016 0.412 hsa-miR-635-001578 0.033 0.461hsa-miR-511-4373236 0.014 0.497

TABLE 14 miRNAs differentially expressed in the putamen of Stage IIIcompared to Stage II (corrected p value < 0.05, fold change > 2 folds)miRNAs p-value Fold change (Stage 3/2) hsa-miR-616-4395525 0.029 2.383hsa-miR-363#-001283 0.036 3.944 hsa-miR-604-001567 0.046 2.567hsa-miR-639-001583 0.004 4.937 hsa-miR-199a-5p-4373272 0.037 0.287hsa-let-7f-1#-002417 0.018 0.161 hsa-miR-1201-002781 0.035 0.371hsa-miR-1301-002827 0.022 0.441 hsa-miR-920-002150 0.040 0.372

TABLE 15 miRNAs differentially expressed in the putamen of Stage IVcompared to Stage II (corrected p value < 0.05, fold change > 2 folds)miRNAs p-value Fold change (Stage 4/2) hsa-miR-1290-002863 0.005 3.942hsa-miR-1248-002870 0.041 2.939 hsa-miR-638-001582 0.005 2.776hsa-miR-1303-002792 0.036 2.440 hsa-miR-296-3p-4395212 0.031 2.412hsa-miR-1255B-002801 0.004 2.314 hsa-miR-378-000567 0.031 2.149hsa-let-7f-1#-002417 0.008 0.136 hsa-miR-1224-3P-002752 0.014 0.210hsa-miR-571-001613 0.041 0.224 mmu-let-7d#-001178 0.037 0.386hsa-miR-1227-002769 0.005 0.486 hsa-miR-511-4373236 0.011 0.497

TABLE 16 miRNAs differentially expressed in the putamen of Stage IVcompared to Stage III (corrected p value < 0.05, fold change > 2 folds)miRNAs p-value Fold change (Stage 4/3) hsa-miR-211-4373088 0.029 50.755hsa-miR-876-3p-4395336 0.003 2.479 hsa-miR-100#-002142 0.005 2.208hsa-miR-1224-3P-002752 0.008 0.175 hsa-miR-639-001583 0.006 0.190hsa-miR-363#-001283 0.040 0.248 hsa-miR-873-4395467 0.019 0.434

We also identified expressions of 29 miRNAs are correlated with theglobal Lewy-type asynucleinopathy (LTS) scores; 32 miRNAs are correlatedwith the degree of depigmentation of the neurons in Substantia Nigra; 33miRNAs are correlated with the worsening of the motor functions, UnifiedPD Rating System (UPDRS) (Table 17-20).

TABLE 17 Disease-correlated miRNAs in the Putamen (p < 0.05) By LTS BySN depigmentation By Score* score UPDRS** Positively correlated 15 20 21Negatively correlated 14 12 16 *LTS score: global Lewy-typeα-synucleinopathy (LTS) scores. **UPDRS: Unified PD Rating Scale.

TABLE 18 miRNAs whose expression levels are correlated with Lewy-typea-synucleinopathy (LTS) scores (p < 0.05) miRNAs r hsa-miR-132-43731430.505 hsa-miR-212-4373087 0.453 hsa-miR-383-4373018 0.449hsa-miR-885-5p-4395407 0.425 hsa-miR-132#-002132 0.406hsa-miR-608-001571 0.401 hsa-miR-183#-002270 0.391 hsa-miR-587-0015400.375 hsa-miR-1269-002789 0.375 hsa-miR-629-001562 0.370hsa-let-7c-4373167 0.350 hsa-miR-559-001527 0.349 hsa-miR-129-3p-43732970.345 hsa-miR-130b#-002114 0.344 hsa-miR-125a-5p-4395309 0.322hsa-miR-574-3p-4395460 −0.317 hsa-miR-374a-4373028 −0.320hsa-miR-720-002895 −0.322 hsa-miR-1275-002840 −0.323hsa-miR-142-5p-4395359 −0.330 hsa-miR-124-4373295 −0.330hsa-miR-26b#-002444 −0.331 hsa-miR-1226#-002758 −0.350hsa-miR-374b#-002391 −0.350 hsa-miR-193a-3p-4395361 −0.364hsa-miR-590-5p-4395176 −0.379 hsa-miR-660-4380925 −0.388hsa-miR-339-5p-4395368 −0.458 hsa-miR-106b#-002380 −0.473

TABLE 19 miRNAs whose expression levels are correlated with SNdepigmentation scores (p < 0.05) miRNAs r hsa-miR-132-4373143 0.548hsa-miR-212-4373087 0.444 hsa-miR-1269-002789 0.410hsa-miR-455-5p-4378098 0.377 hsa-miR-500-4395539 0.376hsa-miR-139-3p-4395424 0.371 hsa-miR-183#-002270 0.366hsa-miR-518b-4373246 0.361 hsa-miR-455-3p-4395355 0.348hsa-miR-629-001562 0.341 hsa-miR-132#-002132 0.340 hsa-miR-23a-43730740.337 hsa-miR-335-4373045 0.320 hsa-miR-580-001621 0.315hsa-miR-383-4373018 0.312 hsa-miR-608-001571 0.312 hsa-miR-548l-0029090.311 hsa-miR-505#-002087 0.299 hsa-let-7c-4373167 0.297hsa-miR-129-3p-4373297 0.291 hsa-miR-17-4395419 −0.314hsa-miR-503-4373228 −0.315 hsa-miR-193a-3p-435361 −0.322hsa-miR-339-5p-4395368 −0.327 hsa-miR-140-5p-4373374 −0.327hsa-miR-574-3p-4395460 −0.347 hsa-miR-302a-4378070 −0.361hsa-miR-10b#-002315 −0.366 hsa-miR-93-4373302 −0.374hsa-miR-590-5p-4395176 −0.419 hsa-miR-106b#-002380 −0.461hsa-miR-124-4373295 −0.500

TABLE 20 miRNAs whose expression levels are correlated with UPDRS (p <0.05) miRNAs r hsa-miR-198-4395384 0.582 hsa-miR-886-3p-4395305 0.528hsa-miR-450a-4395414 0.525 hsa-miR-593-001547 0.523 hsa-miR-920-0021500.518 hsa-miR-521-4373259 0.507 hsa-miR-629-4395547 0.501hsa-miR-517#-001113 0.469 hsa-miR-520c-3p-002400 0.461hsa-miR-125b-2#-002158 0.461 hsa-miR-27a#-002445 0.456hsa-miR-559-001527 0.447 hsa-miR-589-001543 0.442 hsa-miR-886-5p-43953040.430 hsa-miR-1282-002803 0.430 hsa-miR-1270-002807 0.423hsa-miR-99a#-002141 0.422 hsa-miR-181a-2#-002317 0.422hsa-miR-608-001571 0.380 hsa-miR-211-4373088 0.378 hsa-miR-500-0010460.376 hsa-miR-758-4395180 −0.408 hsa-miR-144-002676 −0.415hsa-miR-138-4395395 −0.415 hsa-miR-92a-1#-002137 −0.422hsa-miR-411-4381013 −0.425 hsa-miR-448-4373206 −0.427hsa-miR-324-5p-4373052 −0.432 hsa-miR-203-4373095 −0.439hsa-miR-107-4373154 −0.440 hsa-miR-488-4395468 −0.462hsa-miR-137-4373301 −0.469 hsa-miR-148a-4373130 −0.475hsa-miR-296-5p-4373066 −0.477 hsa-miR-218-4373081 −0.479hsa-miR-153-4373305 −0.507 hsa-miR-552-001520 −0.521

These miRNAs may be involved in the pathological changes in the putamen,contributing to the pathogenesis of Lewy body diseases, including PD;and therefore, they may be new therapeutic targets for treatment of PDat different stages of the diseases.

Example 2 CSF miRNAs as Diagnostic Biomarkers for Presymptomatic andEarly PD, and Cognitive Impairment in PD

Among the differentially expressed miRNAs in the CSF, we identified 80miRNAs that are differentially expressed in ILBD, PD and PDD, whencompared to normal controls (Tables 21-27; FIG. 3); and 87 miRNAs thatare differentially expression in Stages 2, 3, and 4 of Lewy Bodydiseases when compared to normal controls (Table 28-34; FIG. 4).

TABLE 21 miRNAs Differentially Expressed in CSF Among DifferentDiagnostic Groups (p < 0.05; at least 2 fold change). (80 in total)Diagnosis Control ILBD PD PDDnoAD Control 35 (25, 10) 27 (15, 12) 15(13, 2) ILBD 35 (25, 10) 11 (5, 6) 14 (8, 6) PD 27 (15, 12) 11 (5, 6) 20(8, 12) PDDnoAD 15 (13, 2) 14 (8, 6) 20 (8, 12) *Numbers in parenthesesare numbers of (upregulated, downregulated) miRNAs.

TABLE 22 CSF miRNAs differentially expressed in ILBD compared to control(corrected p value < 0.05, fold change > 2 folds) miRNAs p-value FoldChange (ILBD/ctl) hsa-miR-497-001043 0.008 5.645 hsa-miR-126#-0004510.026 5.413 hsa-miR-9#-002231 0.005 5.231 hsa-miR-1300-002902 0.0235.129 hsa-miR-143-4395360 0.007 4.804 hsa-miR-374b-4381045 0.027 4.747hsa-miR-660-4380925 0.005 4.465 hsa-miR-432-001026 0.046 4.313hsa-miR-31#-002113 0.012 3.647 hsa-miR-324-5p-4373052 0.007 3.367hsa-miR-99a#-002141 0.024 3.323 hsa-miR-628-3p-002434 0.002 3.318hsa-miR-652-4395463 0.035 3.011 hsa-miR-1271-002779 0.039 2.775hsa-miR-26b#-002444 0.016 2.706 hsa-miR-195-4373105 0.000 2.680hsa-miR-340-4395369 0.031 2.568 hsa-miR-213-000516 0.032 2.524hsa-miR-29a-4395223 0.003 2.483 hsa-miR-449b-4381011 0.045 2.348hsa-miR-708-4395452 0.021 2.292 hsa-miR-29c-4395171 0.006 2.248hsa-miR-218-4373081 0.016 2.223 hsa-miR-488-001106 0.014 2.155hsa-miR-30e-3p-000422 0.035 2.154 hsa-miR-516-3p-001149 0.040 0.068hsa-miR-296-5p-4373066 0.048 0.252 hsa-miR-671-3p-4395433 0.021 0.274hsa-miR-145-4395389 0.016 0.289 hsa-miR-597-4380960 0.012 0.309hsa-miR-328-4373049 0.025 0.362 hsa-miR-197-4373102 0.039 0.382hsa-miR-92a-4395169 0.019 0.405 hsa-miR-484-4381032 0.023 0.432hsa-miR-700-002895 0.044 0.449

TABLE 23 CSF miRNAs differentialiy expressed in PD compared to control(corrected p value < 0.05, fold change > 2 folds) miRNAs p-value FoldChange (PD/ctl) hsa-miR-664-002897 0.001 18.334 hsa-miR-590-3P-0026770.000 5.568 hsa-miR-577-002675 0.033 4.923 hsa-miR-374b-4381045 0.0324.850 hsa-miR-9#-002231 0.006 4.755 hsa-miR-213-000516 0.001 4.217rno-miR-7#-001338 0.044 3.712 hsa-miR-340#-002259 0.033 3.561hsa-miR-497-001043 0.050 3.504 hsa-miR-454-4395434 0.013 3.093hsa-miR-99a#-002141 0.049 3.050 hsa-miR-191#-002678 0.005 2.907hsa-miR-1183-002841 0.038 2.387 hsa-miR-29a#-002447 0.030 2.243hsa-miR-148b#-002160 0.014 2.137 hsa-miR-142-3p-4373136 0.029 0.260hsa-miR-519a-4395526 0.016 0.318 hsa-miR-511-4373236 0.043 0.319hsa-miR-486-5p-4378096 0.012 0.428 hsa-miR-92a-4395169 0.046 0.435hsa-miR-381-4373020 0.037 0.435 hsa-miR-545-4395378 0.010 0.439hsa-miR-196b-4395326 0.007 0.458 hsa-miR-10b-4395329 0.007 0.463hsa-miR-98-4373009 0.006 0.465 hsa-miR-18a-4395533 0.003 0.470hsa-miR-654-3p-4395350 0.008 0.473

TABLE 24 CSF miRNAs differentially expressed in PDDnoAD compared tocontrol (corrected p value < 0.05, fold change > 2 folds) miRNAs p-valueFold Change (PDDnoAD/ctl) hsa-miR-1300-002902 0.001 10.044hsa-miR-664-002897 0.040 6.381 hsa-miR-374b-4381045 0.011 5.426hsa-miR-1298-002861 0.033 4.569 hsa-miR-135b-4395372 0.003 3.592hsa-miR-363-4378090 0.002 3.357 hsa-miR-660-4380925 0.032 3.189hsa-miR-9#-002231 0.029 2.793 hsa-miR-362-5p-4378092 0.033 2.287hsa-miR-29a-4395223 0.005 2.209 hsa-miR-1264-002799 0.028 2.090hsa-miR-29a#-002447 0.049 2.073 hsa-miR-29c-4395171 0.003 2.043hsa-miR-146b-5p-4373178 0.010 0.430 hsa-miR-518a-3p-4395508 0.020 0.372

TABLE 25 CSF miRNAs differentially expressed in PD compared to ILBD(corrected p value < 0.05, fold change > 2 folds) miRNAs p-value FoldChange (PD/ILBD) hsa-miR-664-002897 0.028 4.750 hsa-miR-720-002895 0.0063.384 hsa-miR-590-3P-002677 0.028 2.431 hsa-miR-625#-002432 0.033 2.096hsa-miR-1183-002841 0.032 2.087 hsa-miR-143-4395360 0.026 0.315hsa-miR-519a-4395526 0.005 0.334 hsa-miR-130a-4373145 0.025 0.414hsa-miR-130b-4373144 0.037 0.422 hsa-miR-500-4395539 0.041 0.424hsa-miR-145#-002149 0.036 0.426

TABLE 26 CSF miRNAs differentially expressed in PDDnoAD compared to ILBD(corrected p value < 0.05, fold change > 2 folds) miRNAs p-value FoldChange (PDDnoAD/ILBD) hsa-miR-886-5p-4395304 0.024 4.734hsa-miR-886-3p-4395305 0.022 4.189 hsa-miR-125a-3p-4395310 0.011 2.761hsa-miR-222#-002097 0.040 2.635 hsa-miR-362-5p-4378092 0.006 2.392hsa-miR-105-4395278 0.048 2.358 hsa-miR-363-4378090 0.036 2.203hsa-let-7d-4395394 0.032 2.128 hsa-miR-143-4395360 0.011 0.329hsa-miR-543-002376 0.034 0.430 hsa-miR-628-3p-002434 0.029 0.441hsa-miR-26b#-002444 0.011 0.460 hsa-miR-146b-5p-4373178 0.035 0.494hsa-miR-376c-4395233 0.035 0.497

TABLE 27 CSF miRNAs differentially expressed in PDDnoAD compared to PD(corrected p value < 0.05, fold change > 2 folds) miRNAs p-value FoldChange (PDDnoAD/PD) hsa-miR-148b-4373129 0.005 4.709hsa-miR-886-5p-4395304 0.013 3.754 hsa-miR-500-4395539 0.006 3.002hsa-miR-886-3p-4395305 0.032 2.889 hsa-miR-519a-4395526 0.045 2.654hsa-miR-511-4373236 0.035 2.279 hsa-miR-363-4378090 0.037 2.114hsa-miR-99b-4373007 0.010 2.038 dme-miR-7-000268 0.015 0.118hsa-miR-1233-002768 0.027 0.229 hsa-miR-409-3p-002332 0.034 0.248hsa-miR-206-000510 0.027 0.263 hsa-miR-590-3P-002677 0.001 0.275hsa-miR-213-000516 0.006 0.316 hsa-miR-629-001562 0.041 0.357hsa-miR-539-4378103 0.037 0.385 hsa-miR-543-002376 0.004 0.407hsa-miR-720-002895 0.014 0.438 hsa-miR-146b-5p-4373178 0.022 0.453hsa-miR-136#-002100 0.013 0.465

TABLE 28 miRNAs Differentially Expressed in CSF Among Different Stagesby the Unified Staging System for LBD (p < 0.05; at least 2 fold change)(87 in total). Stages 0 2 3 4 0 22 (18, 4) 70 (46, 24) 9 (5, 4) 2 22(18, 4) 20 (15, 5) 2 (0, 2) 3 70 (46, 24) 20 (15, 5) 29 (7, 22) 4 9 (5,4) 2 (0, 2) 29 (7, 22) *Numbers in parentheses: numbers of (upregulated,downregulated) miRNAs.

TABLE 29 CSF miRNAs differentially expressed in Stage II compared tocontrols (corrected p values < 0.05, fold change > 2 folds) miRNAsp-value Fold change (Stage 2/0) hsa-miR-664-002897 0.035 7.145hsa-miR-126#-000451 0.041 6.557 hsa-miR-374b-4381045 0.023 5.102hsa-miR-31-4395390 0.007 4.549 hsa-miR-1300-002902 0.032 3.927hsa-miR-577-002675 0.032 3.690 hsa-miR-9#-002231 0.006 3.655hsa-miR-497-001043 0.047 3.493 hsa-miR-660-4380925 0.025 3.394hsa-miR-652-4395463 0.018 3.212 hsa-miR-31#-002113 0.032 2.830hsa-miR-340-4395369 0.037 2.768 hsa-miR-200b-4395362 0.034 2.719hsa-miR-26b#-002444 0.034 2.437 hsa-miR-135b-4395372 0.049 2.391hsa-miR-195-4373105 0.003 2.243 hsa-miR-628-3p-002434 0.027 2.081hsa-miR-29a#-002447 0.048 2.002 hsa-miR-671-3p-4395433 0.023 0.282hsa-miR-142-3p-4373136 0.038 0.348 hsa-miR-145-4395389 0.043 0.407hsa-miR-597-4380960 0.047 0.408

TABLE 30 CSF miRNAs differentiallly expressed in Stage III compared tocontrols (corrected p value < 0.05, fold change > 2 folds) miRNAsp-value Fold change (Stage 3/0) hsa-miR-661-001606 0.008 35.493hsa-miR-1300-002902 0.002 14.271 hsa-miR-497-001043 0.000 10.198hsa-miR-664-002897 0.023 7.823 hsa-miR-577-002675 0.013 7.754hsa-miR-9#-002231 0.003 7.611 hsa-miR-769-5p-001998 0.020 6.804hsa-miR-374b-4381045 0.014 5.960 hsa-miR-31#-002113 0.004 5.909hsa-miR-99a#-002141 0.002 5.850 hsa-miR-638-001582 0.014 5.810hsa-miR-660-4380925 0.005 5.749 hsa-miR-1298-002861 0.037 5.389hsa-miR-592-001546 0.018 4.821 rno-miR-29c#-001818 0.011 4.648hsa-miR-324-5p-4373052 0.001 4.592 hsa-miR-590-3P-002677 0.001 4.436hsa-miR-213-000516 0.004 4.032 hsa-miR-1271-002779 0.005 3.965hsa-miR-9-4373285 0.034 3.796 hsa-miR-622-001553 0.017 3.549hsa-miR-340#-002259 0.037 3.533 hsa-miR-151-5P-002642 0.035 3.373hsa-miR-628-3p-002434 0.006 3.265 hsa-miR-29a-4395223 0.001 3.218hsa-miR-143-4395360 0.046 3.190 hsa-miR-135b-4395372 0.013 3.045hsa-miR-652-4395463 0.031 2.942 hsa-miR-129-3p-4373297 0.045 2.926hsa-miR-218-4373081 0.004 2.880 hsa-miR-454-4395434 0.034 2.870hsa-miR-340-4395369 0.028 2.834 hsa-miR-29c-4395171 0.001 2.802hsa-miR-1226#-002758 0.030 2.670 hsa-miR-30a-3p-000416 0.012 2.617hsa-miR-639-001583 0.042 2.574 hsa-miR-195-4373105 0.000 2.533hsa-miR-191#-002678 0.007 2.389 hsa-miR-29a#-002447 0.030 2.254hsa-miR-148b#-002160 0.020 2.173 hsa-miR-192-4373108 0.028 2.137hsa-miR-148a-4373130 0.003 2.135 hsa-miR-548l-002909 0.036 2.055hsa-miR-1264-002799 0.004 2.052 hsa-miR-19b-4373098 0.014 2.013hsa-miR-27a#-002445 0.021 2.007 hsa-miR-483-3p-002339 0.005 0.081hsa-miR-1260-002896 0.026 0.122 hsa-miR-296-5p-4373066 0.019 0.140hsa-miR-145-4395389 0.001 0.161 hsa-miR-671-3p-4395433 0.009 0.166hsa-miR-125a-5p-4395309 0.011 0.243 hsa-miR-197-4373102 0.005 0.257hsa-miR-92a-4395169 0.002 0.284 hsa-miR-597-4380960 0.022 0.292hsa-miR-328-4373049 0.001 0.305 hsa-miR-484-4381032 0.002 0.319hsa-miR-125b-4373148 0.009 0.363 hsa-miR-345-4395297 0.019 0.413hsa-miR-10b-4395329 0.008 0.461 hsa-miR-365-4373194 0.039 0.463hsa-miR-98-4373009 0.008 0.464 hsa-miR-545-4395378 0.020 0.468hsa-miR-28-3p-4395557 0.000 0.469 hsa-miR-25-4373071 0.006 0.472hsa-miR-654-3p-4395350 0.009 0.474 hsa-miR-486-5p-4378096 0.033 0.475hsa-miR-196b-4395326 0.014 0.482 hsa-miR-193a-5p-4395392 0.050 0.486hsa-miR-199a-5p-4373272 0.040 0.497

TABLE 31 CSF miRNAs differentially expressed in Stage IV compared tocontrols (corrected p value < 0.05, fold change > 2 folds) miRNAsp-value Fold change (Stage 4/0) hsa-miR-664-002897 0.0267 6.6111hsa-miR-374b-4381045 0.0298 4.2124 hsa-miR-1300-002902 0.0443 3.9213hsa-miR-9#-002231 0.0246 2.8799 hsa-miR-191#-002678 0.0377 2.2135hsa-miR-142-3p-4373136 0.0103 0.2291 hsa-miR-197-4373102 0.0326 0.4014hsa-miR-518a-3p-4395508 0.0247 0.4188 hsa-miR-320B-002844 0.0210 0.4882

TABLE 32 CSF miRNAs differentially expressed in Stage III compared toStage II (corrected p value < 0.05, fold change > 2 folds) miRNAsp-value Fold change (Stage 3/2) hsa-miR-661-001606 0.014 10.926hsa-miR-638-001582 0.018 4.388 hsa-miR-144#-002148 0.002 4.147hsa-miR-566-001533 0.010 3.230 hsa-miR-622-001553 0.022 3.205hsa-miR-497-001043 0.031 2.919 hsa-miR-663B-002857 0.024 2.748hsa-miR-1226#-002758 0.023 2.555 hsa-miR-30a-3p-000416 0.002 2.417hsa-miR-324-5p-4373052 0.040 2.290 hsa-miR-590-3P-002677 0.042 2.244rno-miR-29c#-001818 0.044 2.193 hsa-miR-148a-4373130 0.015 2.140hsa-miR-31#-002113 0.023 2.088 hsa-miR-218-4373081 0.029 2.077hsa-miR-483-3p-002339 0.011 0.142 hsa-miR-125b-4373148 0.001 0.251hsa-miR-125a-5p-4395309 0.013 0.272 hsa-miR-145-4395389 0.015 0.395hsa-miR-130b-4373144 0.047 0.448

TABLE 33 CSF miRNAs differentially expressed in Stage IV compared toStage II (corrected p value < 0.05, fold change > 2 folds) miRNAsp-value Fold change (Stage 4/2) hsa-miR-181a-4373117 0.022 0.436hsa-miR-338-3p-4395363 0.049 0.459

TABLE 34 CSF miRNAs differentially expressed in Stage IV compared toStage III (corrected p value < 0.05, fold change > 2 folds) miRNAsp-value Fold change (Stage 4/3) hsa-miR-483-3p-002339 0.041 5.103hsa-miR-145-4395389 0.001 4.830 hsa-miR-296-5p-4373066 0.033 4.172hsa-miR-125a-5p-4395309 0.010 3.893 hsa-miR-671-3p-4395433 0.022 3.134hsa-miR-125b-4373148 0.001 2.949 hsa-miR-484-4381032 0.014 2.231hsa-miR-661-001606 0.010 0.142 hsa-miR-497-001043 0.007 0.216hsa-miR-1291-002838 0.004 0.240 hsa-miR-638-001582 0.013 0.244hsa-miR-31#-002113 0.004 0.245 hsa-miR-378-002243 0.013 0.267hsa-miR-1271-002779 0.002 0.267 hsa-miR-219-2-3p-4395501 0.033 0.272hsa-miR-622-001553 0.010 0.289 hsa-miR-99a#-002141 0.007 0.294hsa-miR-144#-002148 0.024 0.324 hsa-miR-769-5p-001998 0.037 0.325hsa-miR-6638-002857 0.025 0.361 hsa-miR-1226#-002758 0.011 0.363hsa-miR-142-3p-4373136 0.033 0.379 hsa-miR-151-5P-002642 0.001 0.382hsa-miR-3208-002844 0.000 0.413 hsa-miR-12558-002801 0.044 0.417hsa-miR-660-4380925 0.006 0.439 hsa-miR-192-4373108 0.045 0.479hsa-miR-218-4373081 0.026 0.485 hsa-miR-30a-3p-000416 0.026 0.493

We also identified expressions of 26 miRNAs are correlated with theglobal Lewy-type asynucleinopathy (LTS) scores; 36 miRNAs are correlatedwith the degree of depigmentation of the neurons in Substantia Nigra; 40miRNAs are correlated with the worsening of the motor functions, UnifiedPD Rating System (UPDRS) (Tables 35-38).

TABLE 35 Disease-correlated miRNAs in CSF (p < 0.05) By LTS By SNdepigmentation By Score* score UPDRS** Positively correlated 23 19 9Negatively correlated 3 17 31 *LTS score: global Lewy-typeα-synucleinopathy (LTS) scores. **UPDRS: Unified PD Rating Scale.

TABLE 36 CSF miRNAs whose expression levels are correlated withLewy-type a-synucleinopathy (LTS) scores (p < 0.05) miRNAs rhsa-miR-664-002897 0.519 hsa-miR-148b#-002160 0.496 hsa-miR-29a#-0024470.453 hsa-miR-592-001546 0.403 hsa-miR-363-4378090 0.400hsa-miR-135b-4395372 0.397 hsa-miR-135a-4373140 0.390hsa-miR-1298-002861 0.389 hsa-miR-34a#-002316 0.389 hsa-miR-374b-43810450.384 rno-miR-7#-001338 0.381 hsa-miR-590-3P-002677 0.379rno-miR-29c#-001818 0.371 hsa-miR-1264-002799 0.371 hsa-miR-22#-0023010.358 hsa-miR-30a-3p-000416 0.353 hsa-miR-650-001603 0.346hsa-miR-340-4395369 0.338 hsa-miR-454-4395434 0.336hsa-miR-767-3p-001995 0.333 hsa-miR-101#-002143 0.329hsa-miR-190-4373110 0.328 hsa-miR-213-000516 0.320hsa-miR-486-5p-4378096 −0.373 hsa-miR-196b-4395326 −0.435hsa-miR-518a-3p-4395508 −0.439

TABLE 37 CSF miRNAs whose expression levels are correlated with SNdepigmentation scores (p < 0.05) miRNAs r hsa-miR-363-4378090 0.443hsa-miR-374b-4381045 0.395 hsa-miR-184-4373113 0.339hsa-miR-135b-4395372 0.325 hsa-miR-628-5p-4395544 0.323hsa-miR-374a-4373028 0.309 hsa-miR-331-3p-4373046 0.294hsa-miR-217-4395448 0.289 hsa-miR-590-5p-4395176 0.286hsa-miR-664-002897 0.285 hsa-let-7a-4373169 0.279 hsa-miR-26b-43951670.272 hsa-let-7f-4373164 0.263 hsa-miR-362-5p-4378092 0.263hsa-miR-455-3p-4395355 0.262 hsa-miR-449b-4381011 0.262hsa-miR-99a-4373008 0.255 hsa-miR-135a-4373140 0.254 hsa-miR-26a-43951660.253 hsa-miR-218-2#-002294 −0.227 hsa-miR-139-3p-4395424 −0.232hsa-miR-143#-002146 −0.246 hsa-miR-185-4395382 −0.249 hsa-miR-630-001563−0.255 hsa-miR-519a-4395526 −0.255 hsa-miR-376a#-002127 −0.260hsa-let-7c-4373167 −0.263 hsa-miR-133b-4395358 −0.271hsa-miR-145#-002149 −0.282 hsa-miR-181a-4373117 −0.299hsa-miR-182#-000483 −0.308 hsa-miR-200a#-001011 −0.313hsa-miR-197-4373102 −0.315 hsa-miR-146a#-002163 −0.318hsa-miR-142-3p-4373136 −0.354 hsa-miR-518a-3p-4395508 −0.363

TABLE 38 CSF miRNAs whose expression levels are correlated with UPDRS (p< 0.05) miRNAs r hsa-miR-101#-002143 0.572 hsa-miR-222#-002097 0.565hsa-miR-626-001559 0.555 hsa-miR-29a#-002447 0.503 hsa-miR-1238-0029270.469 hsa-miR-1260-002896 0.453 hsa-miR-363#-001283 0.393hsa-miR-1244-002791 0.388 hsa-miR-567-001534 0.385 hsa-miR-101-4395364−0.230 hsa-miR-218-4373081 −0.233 hsa-miR-27a#-002445 −0.234hsa-miR-330-3p-4373047 −0.236 hsa-miR-30e-3p-000422 −0.237hsa-miR-29b-2#-002166 −0.239 hsa-miR-411-4381013 −0.241hsa-miR-875-5p-002203 −0.242 hsa-miR-337-5p-4395267 −0.244hsa-miR-130a-4373145 −0.245 hsa-miR-454#-001996 −0.257 dme-miR-7-000268−0.261 hsa-miR-19b-1#-002425 −0.263 hsa-miR-32-4395220 −0.263hsa-miR-137-4373301 −0.265 hsa-miR-122-4395356 −0.268hsa-miR-518a-3p-4395508 −0.269 hsa-miR-219-5p-4373080 −0.274hsa-let-7b-4395446 −0.278 hsa-miR-655-4381015 −0.279 hsa-miR-1264-002799−0.282 hsa-miR-1262-002852 −0.289 hsa-miR-518b-4373246 −0.290hsa-miR-152-4395170 −0.292 hsa-miR-338-3p-4395363 −0.294hsa-miR-520D-3P-002743 −0.316 hsa-miR-488-4395468 −0.354hsa-miR-551b-4380945 −0.375 hsa-miR-625-4395542 −0.382hsa-miR-519a-4395526 −0.419 hsa-miR-21-4373090 −0.429

More importantly, we identified a series of miRNA signatures as firstgeneration of diagnostic biomarkers for PD and the progression of PD:

1) miRNA signature composed by 14 miRNAs that distinguishes ILBD tonormal controls, which may be used as a diagnostic biomarker forpre-symptomatic PD (FIG. 5): miR-516, miR-191#, miR-449b, miR-497,miR-628, miR-213, miR-23a, miR-29#, miR-488, miR-597, miR-296-5p,miR-671-3p, miR-654-3p, miR-99#;

2) miRNA signature composed of 13 miRNAs that distinguishes normalcontrol subjects from PD, which may be used as a diagnostic biomarkerfor diagnosis of early PD (FIG. 6): miR-590-3p, miR-213, miR-9#,miR-191#, miR-497, miR-664, miR-99a#, miR-1183, miR-340#, miR-628-3p,miR-7#, miR-29a#, miR-142-3p;

3) miRNA signature composed by 6 miRNAs that distinguishes ILBD from PD,which may change from asymptomatic Lewis Body disease to symptomatic PD,and may be used as a diagnostic biomarker for diagnosis of early PD(FIG. 7): miR-664, miR-1285, miR-1183, miR-143, miR-519a, miR-603;

4) miRNA signature composed by 8 miRNAs that distinguish clinicallynormal subjects (including normal control and ILBD subjects), which mayalso be used early diagnostic biomarkers for PD (FIG. 8): miR-590-3p,miR-664, miR-519a, miR-340*, miR-720, miR-142-3p, miR-185, and miR-213;and

5) miRNA signature composed by 7 miRNAs which distinguish PD from PDwith dementia, and may be used as a diagnostic biomarker for cognitiveimpairment in PD patients (FIG. 9): miR-590-3p, miR-213, miR-409-3p,miR-500, miR-7, miR-206 and miR-629.

Example 3 miR-212 and miR-132 Play Important Roles in Pathogenesis of PDand are Novel Therapeutic Targets for Treatment of PD and L-Dopa-InducedDyskinesia

Among the miRNAs in the putamen, which are correlated with theprogression of disease, miR-212 and miR-132 are highly expressed theputamen, and are significantly increased (˜1.6 fold) in the putamen ofPD cases, compared to controls (FIG. 8). miR-212 and miR-132 areclustered in intron 1 of a highly-conserved, non-coding RNA gene onhuman chromosome 1p13.3, and have high sequence homology with the sameseed sequences, therefore, share most of their downstream targets andfunctions (FIG. 10). Interestingly, mature miR-132 is expressed at amuch higher level than miR-212 in the putamen (>60 fold), suggestingthat miR-132 plays a major role in the putamen.

Correlation study with all cases regardless of their clinical diagnosisshowed that levels of miR-212 and miR-132 expression in the putamen aresignificantly correlated to the global Lewy-type α-synucleinopathy (LTS)scores and the degree of substantia nigra (SN) pigmented neuron lossscore (FIG. 11), with the highest correlation scores among alldifferentially expressed miRNAs, strongly suggesting that expression ofmiR-212 and/or miR-132 correlates with SN neurodegeneration and PDpathology, and that misregulation of miR-212 and/or miR-132 are involvedin PD. Multiple lines of evidence support that miR-212 and/or miR-132play important roles in pathogenesis of PD and are novel therapeutictargets for treatment of PD and L-Dopa-induced dyskinesia:

1) Our target prediction analysis showed that Nurr1, a member of nuclearreceptor superfamily, is a predicted target of miR-212 and/or miR-132.Nurr1 is essential for the differentiation and survival of nigraldopaminergic neurons5-7. Mutations are associated with PD. Therefore,miR-212 and/or miR-132 may regulate the function and survival ofdopaminergic (DA) neurons by modulating Nurr1.

2) Our functional annotation analysis of predicted target genes ofmiR-212 and miR-132 revealed significant enrichment of genes involved inneurotrophin signaling (p=0.01), MAPK signaling (p=0.008) and longtermpotentiation (p=0.02), consistent with reports that miR-212 and miR-132are neurotrophin- and activity-regulated miRNAs and are important forneural plasticity.

3) NMDA receptors (NMDA-Rs) are shown to mediate neurotrophin- andactivity-induced induction of miR-212 and miR-132. In PD, depletion ofnigrostriatal DA results in relative glutamatergic overactivity in thestriatum, which plays central roles in PD as well as L-dopa induceddyskinesia. Therefore, upregulation of miR-212 and/or miR-132 in PDputamen may be a result of loss of dopamine input and relativeoveractivity through NMDA-R;

4) miR-212 and miR-132 are shown to target Methyl CpG-binding protein-2(MeCP2), which promote the expression of brain-derived neurotrophicfactor (BDNF), a potent trophic factor for DA neurons, which has beenshown to be decreased in substantia nigra (SN) and striatum PD patientsand animal models. Therefore, increased of expression of miR-212 and/ormiR-132 may inhibit the expression of MeCP2 and BDNF, and contribute tothe pathogenesis of PD;

5) miR-212 and miR-132 are shown to promote NF-κB- and p53-activationthrough repression of silent information regulator 1 (SIRT1), aNAD+-dependent deacetylase, which deacetylates and inactivates p53 andthe p65 subunit of NF-κB29-32. Increased p53 and NF-κB activation in SNand striatum promote cellular senescence and apoptosis. Therefore,miR-212 and/or miR-132 may contribute to nigrostriatal neurodegenerationthrough modulating NF-κB and p53 activation.

Collectively, these data suggest that upregulation of miR-212 and/ormiR-132 in the putamen may be a result of loss of nigrostriatal DA andoveractivation of NMDA-Rs; miR-212 and/or miR-132 may mediate NMDARoveractivation-induced excitotoxicity and play important roles in thepathogenesis of PD through argeting MeCP2, SIRT1 and Nurr1, modulatingNurr1-, BDNF-, NF-κB- and p53-involved pathogenetic pathways. Therefore,prevention of loss of DA innervation-resulted upregulation of miR-212and/or miR-132 may be neuroprotective for the nigrostriatal system andprevent the development of PD. miR-212 and/or miR-132 may be noveltherapeutic targets for the treatment of PD.

The above Figures and disclosure are intended to be illustrative and notexhaustive. This description will suggest many variations andalternatives to one of ordinary skill in the art. All such variationsand alternatives are intended to be encompassed within the scope of theattached claims. Those familiar with the art may recognize otherequivalents to the specific embodiments described herein whichequivalents are also intended to be encompassed by the attached claims.

I claim:
 1. A method of identifying a subject at risk of developing orhaving Parkinson's Disease (PD), the method comprising: a) isolating abiological sample from the subject; b) detecting a level of expressionin a miRNA signature in the biological sample from the subject, themiRNA signature comprising a plurality of miRNAs; the plurality ofmiRNAs comprising: i) SEQ ID NOs: 204, 110, 192, 200, 244, 2, 134, 144,198, 234, 143, 261, 255, and 179 (miR-516-3p, miR-191#, miR-449b,miR-497, miR-628-3p, miR-213, miR-23a, miR-29a#, miR-488, miR-597,miR-296-5p, miR-671-3p, miR-654-3p, and miR-99a#); ii) SEQ ID NOs: 1-13,(miR-590-3p, miR-213, miR-9#, miR-191#, miR-497, miR-664, miR-99a#,miR-1183, miR-340#, miR-628-3p, miR-7#, miR-29a#, miR-142-3p); iii) SEQID NOs: 14-20 and 2 (miR-590-3p, miR-664, miR-519a, miR-340#, miR-720,miR-142-3p, miR-185, and miR-213); or iv) SEQ ID NOs: 28, 2 and 30-34(miR-590-3p, miR-213, miR-409-3p, miR-500, miR-7, miR-206, and miR-629);c) comparing the level of expression of the plurality of miRNAs in thesample to a level of expression of the plurality of miRNAs in areference, wherein an increased or decreased level of expression in thesample compared to the level of expression in the reference for each ofthe plurality of miRNAs identifies the subject as having PD or who is atrisk of developing PD.
 2. The method of claim 1, wherein the referenceis a sample from a normal, healthy subject.
 3. The method of claim 1,wherein the plurality of miRNAs comprise SEQ ID NOs: 204, 110, 192, 200,244, 2, 134, 144, 198, 234, 143, 261, 255, and 179 (miR-516-3p,miR-191#, miR-449b, miR-497, miR-628-3p, miR-213, miR-23a, miR-29a#,miR-488, miR-597, miR-296-5p, miR-671-3p, miR-654-3p, and miR-99a#) andthe miRNA signature is indicative of pre-symptomatic PD.
 4. The methodof claim 1, wherein the plurality of miRNAs comprise SEQ ID NOs: 1-13,(miR-590-3p, miR-213, miR-9#, miR-191#, miR-497, miR-664, miR-99a#,miR-1183, miR-340#, miR-628-3p, miR-7#, miR-29a#, miR-142-3p) and themiRNA signature is indicative of early PD.
 5. The method of claim 1,wherein the plurality of miRNAs comprise SEQ ID NOs: 14-20 and 2(miR-590-3p, miR-664, miR-519a, miR-340#, miR-720, miR-142-3p, miR-185,and miR-213) and the miRNA signature is indicative of early PD.
 6. Themethod of claim 1, wherein the plurality of miRNAs comprise SEQ ID NOs:28, 2 and 30-34 (miR-590-3p, miR-213, miR-409-3p, miR-500, miR-7,miR-206, and miR-629) and the miRNA signature is indicative of cognitiveimpairment of PD.